The current model for the structure of hyaline cartilage proteoglycan has been elucidated, in large part, by examining the fragments produced by the action of trypsin on this macromolecule. Cyanogen bromide (CNBr) has been widely used as a protein-cleavage reagent in structural studies but has been used with proteoglycans to a limited extent. CNBr quantitatively and specifically breaks the peptide chain at methionine residues. Since the methionine content of proteins is relatively low, reaction with CNBr produces a limited number of peptides. Preliminary studies in this laboratory indicate that the reaction of CNBr with cartilage proteoglycans yields discrete monodisperse peptides. In this study, it is proposed to examine the CNBr-cleavage of rat chondrosarcoma proteoglycan. The proteoglycan will be isolated under conditions found to minimize proteolytic degradation. To increase the homogeneity of the molecule, the chondroitin sulfate chains will be removed from the proteoglycan with chondroitinase and the resultant protein core will be purified by affinity chromatography on immobilized hyaluronic acid. After digestion of the core with CNBr, the CNBr peptides will be purified and analyzed for their amino acid and carbohydrate contents. Finally, the amino acid sequences of the homogeneous peptides will be at least partially determined. Antisera will be raised in rabbits against whole proteoglycan, protein core and trypsin-generated fragments. The reactivity of these antisera with the CNBr-peptides will be determined as a means of identifying the antigenically active sites of proteoglycans. The proposed investigations will aid in the further elucidation of the structural and immunological properties of proteoglycans and in identifying associated proteoglycan and collagen peptides in CNBr-digested cartilage. Additionally, the methods developed in this study will be useful for the analysis of proteoglycans from healthy and diseased articular cartilage. If proteoglycans are partially degraded in the disease state, one or more of the CNBr peptides will be altered or missing, thus pinpointing the site of initial proteoglycan breakdown.